Highly pure neurotoxic component of a botulinum toxin, process for preparing same, and uses thereof

ABSTRACT

The present invention relates to a process for preparing a highly pure neurotoxic component of a  botulinum  toxin by cultivating  clostridium botulinum  under conditions that allow production of a  botulinum  toxin, and isolating the neurotoxic component from the  botulinum  toxin. In addition, the present invention relates to a highly pure neurotoxic component of a  botulinum  toxin obtainable by the process of the present invention and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a § 371 National Stage Application of PCTInternational Application Number PCT/EP2014/00289 filed on 30 Jul. 2014which claims priority to EP 13 003 792.2 filed 30 Jul. 2013.

BACKGROUND Field of the Invention

The present invention relates to a process for preparing a highly pureneurotoxic component of a botulinum toxin by cultivating Clostridiumbotulinum under conditions that allow production of a botulinum toxin,and isolating the neurotoxic component from the botulinum toxin. Inaddition, the present invention relates to a highly pure neurotoxiccomponent of a botulinum toxin obtainable by the process of the presentinvention and uses thereof.

Description of Related Art

Botulinum toxins are the most potent protein toxins for humans. They actby blocking acetylcholine release at the neuromuscular junctionresulting in denervation of muscles. Botulinum toxins also have activityat other peripheral cholinergic nerve terminals and lead, for example,to reduced salivation or sweating and to diminished facial lines andwrinkles. Due to their specificity of mode of action, the range ofclinical applications of botulinum toxins is continuously growing, andbotulinum toxins are today being used extensively as pharmaco-cosmetics.

The botulinum toxins are synthesized and released by certain Clostridiumspp. in the form of large complexes comprising the botulinum toxinmolecule (the “neurotoxic component”) and associated non-toxic bacterialproteins (also referred to as “complexing proteins”). The complexingproteins include different non-toxic hemagglutinin (HA) proteins andnon-toxic non-hemagglutinin (NTNH) proteins. The molecular weight of thetoxin complex varies among the seven distinct botulinum toxin serotypes(A, B, C, D, E, F and G) from about 300 kDa to about 900 kDa. Thecomplexing proteins provide stability to the neurotoxic component.Unlike the toxin complex, the neurotoxic component in its isolated andpure form, i.e. devoid of any complexing clostridial proteins, is acidlabile and does not resist the aggressive environment in thegastrointestinal tract.

The neurotoxic component is synthesized as an inactive single-chainprecursor (non-cleaved polypeptide) having a molecular weight, for allseven of the known botulinum toxin serotypes, of about 150 kDa. Thissingle-chain precursor is activated by proteolytic cleavage to generatea disulfide-linked two-chain protein. The 50 kDa light chain containsthe catalytic domain, whereas the 100 kDa heavy chain contains aninternal translocation domain and a receptor binding domain. The 100 kDaheavy chain mediates binding to pre-synaptic cholinergic nerve terminalsand internalization of the toxin into the cell. The 50 kDa light chainis responsible for the toxic effects, acting as zinc-endopeptidase andcleaving specific proteins responsible for membrane fusion (proteins ofthe SNARE complex). By disrupting the process of membrane fusion withinthe nerve cells, botulinum toxins prevent the release of acetylcholineinto the synaptic cleft.

The botulinum toxin serotype A complex (BoNT/A-complex) was firstapproved for human use in the United States in 1989 for the treatment ofstrabism, blepharospasm, and other disorders. Today, the “A” form of thebotulinum toxin complex is available commercially from several sources,for example from Allergan under the trade name Botox®, from Ipsen underthe trade name Dysport®, and from Galderma under the trade nameAzzalure®.

However, in a significant number of cases, patients producedneutralizing antibodies in response to repeated BoNT/A-complexinjections. It is believed that this effect is associated with thecomplexing proteins of the BoNT/A-complex. The patients affected becomeso-called “secondary non-responders”, and therapy with theBoNT/A-complex is no longer effective. This risk for suchantibody-induced therapy failure was found to affect no less than 10% to20% of the subjects treated. Another disadvantage associated with theuse of the botulinum toxin complex is its regional or systemic spreadfollowing injections into the target muscles. For example, studies usingsingle-fibre electromyography (SF-EMG) have shown increased jitter inmuscles distant from the injection site.

These disadvantages are not observed with the administration of the pureneurotoxic component. In particular, administration of the pureneurotoxic component reduces the risk of non- or decreased response,which is of particular importance for patients undergoing long-termtreatments. Other benefits associated with the pure neurotoxic componentinclude a fast onset of effect and an excellent temperature stability,which even obviates the need for a cold chain and storage in arefrigerator. A formulation containing only the neurotoxic component oftype A without any complexing proteins is commercially available fromMerz under the trade name Xeomin® and Boconture®.

The neurotoxic component can be prepared by cultivating botulinum toxinproducing clostridial strains and isolating the neurotoxic componentfrom the produced botulinum toxin complex through a series ofprecipitation and chromatographic steps. If naturally occurringclostridial strains are used, the botulinum toxin is produced andreleased by clostridial bacteria in its active, acutely toxic form.Therefore, specific measures must be taken to avoid unfavorable healthconsequences to the personnel concerned with the production of thebotulinum toxin and/or the purification of the neurotoxic component fromthe toxin complex. In order to reduce the risk of exposure to toxicaerosols, WO 2006/133818 A1 proposes, for example, to conduct theproduction of botulinum toxins in an isolator operated at a lowerpressure than that of the surrounding production room to avoid contactof the operator with any toxic material.

While the manufacturing process described in WO 2006/133818 A1 ensuresadequate operational safety, it still leaves room for improvements inthe purity of the neurotoxic component produced. In the pharmaceuticalindustry, a high chemical and microbial purity is critically important.Therefore, the ultimate goal of drug developers is to achieve drugpurity as high as possible to establish the desired safety and efficacy.

Accordingly, the objective of the present invention is to provide animproved process for preparing a highly pure neurotoxic component of abotulinum toxin in a safe manner.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a process forpreparing a highly pure neurotoxic component of a botulinum toxin, theprocess comprising the steps of:

-   -   (a) cultivating Clostridium botulinum under conditions that        allow production of a botulinum toxin, and    -   (b) isolating the neurotoxic component from the botulinum toxin,        wherein cultivating step (a) and isolating step (b) are        conducted in a pressure gradient device, which comprises a first        isolator unit containing a fermenter for cultivating Clostridium        botulinum and, optionally, a second or further isolator unit as        well as a safety work bench which is a Class II BSC (Biological        Safety Cabinet) provided with a transfer system that allows for        the aseptic transfer of material into and out of the BSC.

The first and second or further isolator units are located in aproduction room that is connected to the environment via an air lock,wherein the pressure in the first and second or further isolator unitsis lower than that in the production room, the pressure in theproduction room is lower than ambient pressure, and the pressure in theair lock is higher than ambient pressure. The safety work bench is alsolocated in the production room.

In another aspect, the present invention provides a highly pureneurotoxic component of a Clostridium botulinum toxin having asingle-chain content of less than 2.00 wt. %.

In yet another aspect, the present invention provides a pharmaceuticalcomposition comprising a highly pure neurotoxic component of aClostridium botulinum toxin as described herein and one or morepharmaceutically acceptable carriers.

In a yet further aspect, the present invention provides a highly pureneurotoxic component of a Clostridium botulinum toxin as describedherein for use as a medicament.

In still another aspect, the present invention provides a highly pureneurotoxic component of a Clostridium botulinum toxin as describedherein for use in the treatment of a disease associated with hyperactivecholinergic innervation of muscles or exocrine glands.

Preferred embodiments of the present invention are set forth in theappended dependent claims.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

It has been surprisingly found that there are key production parametersthat have not been considered so far but which can exert a profoundinfluence upon the quality, in particular upon the purity, of aneurotoxic component of a botulinum toxin. Furthermore, the productionprocess of the present invention meets the legal requirements pertainingto safety, health and the environment, and fosters a safe andnon-hazardous work environment. In other words, the present invention isbased on the unexpected finding that additional modes of operation ofthe manufacturing process exist, which are not only safe with respect toenvironmental and human health issues but also provide a neurotoxiccomponent of a botulinum toxin of superior quality, in particularsuperior quality.

In a first aspect, the present invention relates to a process forpreparing a highly pure neurotoxic component of a botulinum toxin, theprocess comprising the steps of:

-   -   (a) cultivating Clostridium botulinum under conditions that        allow production of a botulinum toxin, and    -   (b) isolating the neurotoxic component from the botulinum toxin.

As used herein, the terms “botulinum toxin” or “botulinum toxin complex”are interchangeable and refer to a high molecular weight complexcomprising the neurotoxic component of approximately 150 kDa and, inaddition, non-toxic proteins of Clostridium spp., includinghemagglutinin and non-hemagglutinin proteins. Also, the terms “botulinumtoxin” and “botulinum toxin complex” are intended to cover all seventoxin serotypes (i.e. serotypes A, B, C, D, E, F and G) as well assubtypes thereof (e.g., A1, A2, C1, C2, etc.).

The term “neurotoxic component”, as used herein, refers to the botulinumtoxin protein molecule included in the botulinum toxin complex (alsoreferred to as the “pure toxin” or the “pure neurotoxin”). In otherwords, a “neurotoxic component” within the meaning of the presentinvention is not associated with and devoid of any associated non-toxinproteins of Clostridium botulinum, including hemagglutinin andnon-hemagglutinin proteins. Preferably, it is also free of RNApotentially associated with the neurotoxic component.

It is further pointed out that a “neurotoxic component” within themeaning of the present invention encompasses the single-chain precursorprotein of approximately 150 kDa and the proteolytically processeddi-chain form of the neurotoxic component, comprising the light chain(LC) of approximately 50 kDa and the heavy chain (HC) of approximately100 kDa, which are commonly linked by one or more disulfide bonds. Thoseof skill in the art will appreciate that full biological activity isattained only after proteolytic activation, even though it isconceivable that the unprocessed precursor can exert some biologicalfunctions. “Biological function” may refer to (a) receptor binding, (b)internalization, (c) translocation across the endosomal membrane intothe cytosol, and/or (d) endoproteolytic cleavage of proteins involved insynaptic vesicle membrane fusion.

Preferably, the neurotoxic component is derived from a naturallyoccurring botulinum toxin complex of serotype A, B, C, D, E, F or G. Aparticularly preferred neurotoxic component within the context of thepresent invention is derived from Clostridium botulinum toxin serotypeA, in particular from the Clostridium botulinum toxin type A produced bythe Hall strain (ATCC 3502). However, within the context of the presentinvention, a neurotoxic component may also be a recombinantly producedneurotoxic component, including a chimeric (fused) neurotoxic component.Also included are genetically modified neurotoxic components containing1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 20 amino acid mutations. Amutation may be a substitution, an insertion or a deletion. In addition,neurotoxic components which contain chemically modified amino acids, forexample one or more amino acids that are glycosylated, acetylated,lipidated or otherwise modified, are also comprised within the term“neurotoxic component”.

The term “highly pure neurotoxic component” within the meaning of thepresent invention refers to a purified neurotoxic component, or acomposition, preparation or formulation thereof, which essentiallycontains no other solid ingredients, and which may be prepared by theprocess described in detail herein. Furthermore, the term “highly pure”,as used herein, refers to a neurotoxic component of a botulinum toxin,or a formulation, preparation or composition thereof that is free ofcomplexing proteins (product-related impurities), other clostridialproteins (non-product related impurities), and non-clostridial proteins.Preferably, the term “highly pure” refers to a total purity of at least99.90 wt. %, more preferably at least 99.95 wt. %, and most preferablyat least 99.99 wt. %. “Total purity” means the weight percentage of thesingle-chain and two-chain forms of a neurotoxic component, based on thetotal weight of a sample of the highly pure neurotoxic component of thepresent invention. In accordance with the present invention, the purityis assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).

In step (a), Clostridium botulinum is cultivated (or fermented) in asuitable fermentation medium, such as a medium containing 2% proteosepeptone, 1% yeast extract, 1% glucose and 0.05% sodium thioglycolate, inorder to produce a botulinum toxin. As used herein, the term“cultivating” is interchangeably used with the term “fermenting”. Theterm “Clostridium botulinum” is intended to include Clostridiumbotulinum type A, B, C, D, E, F or G. Within the context of the presentinvention, a Clostridium botulinum strain producing a botulinum toxin oftype A, i.e. a Clostridium botulinum type A, is preferably used.Particularly preferred for use herein is Clostridium botulinum type AHall strain (ATCC 3502) (in the following referred to as “the Hallstrain”). Processes for cultivating Clostridium botulinum to produce thetoxin complex are known in the art (see, e.g., Schantz E. and KautterD., J. Assoc. Off. Anal. Chem. 61:96-99, 1978).

The fermentation (cultivation) temperature in step (a) of the process ofthe present invention depends on the specific Clostridium botulinumstrain and fermentation conditions used. Preferably, the temperature isconstantly maintained in a pre-defined, narrow temperature range. Forexample, for the production of Clostridium botulinum toxin type A, inparticular the Hall strain, the fermentation temperature is preferablyset to and maintained at 33.5° C.±1.0° C., more preferably 33.5° C.±0.5°C., and most preferably 33.5° C.±0.2° C.

It was found by the present inventors that a constant temperature ofabout 33.5° C. is the optimal temperature for botulinum toxinproduction. Temperatures that are too high, too low and/or too variablewill cause the Clostridium botulinum to produce undesirable compounds.Surprisingly, it was found that, if the fermentation temperature is notcontrolled to be within the temperature ranges indicated above, theamount of the unwanted inactive single-chain form of the neurotoxiccomponent significantly increases. It is stressed that, within thecontext of the present invention, the single-chain form of theneurotoxic component is regarded as being an undesirable “impurity”since it is not proteolytically processed and essentially inactive.

In line with the present invention, the fermentation temperature ispreferably set to and maintained at the indicated temperature rangeusing a heating jacket. As used herein, a “heating jacket” is a materialthat surrounds large surface areas, typically the entire side walls, ofa fermenter and can be heated. For example, a heating jacket mayincorporate different layers to provide the temperature stabilityrequired for the fermentation, such as a thermoelectric base layer forheating, an insulation layer to avoid temperature loss, and a waterproofouter layer for protection of the heating jacket from the hazards of thefermentation environment. In contrast to increasing and maintaining thetemperature by means of a heating rod, the use of a heating jacketessentially avoids heating differences among different sites in theculture medium, even if the culture medium is not stirred as istypically the case for the process of the present invention, therebyensuring a uniform temperature distribution.

The growth of the clostridial cultures during fermentation (i.e. thecell density) is preferably assessed by the turbidity of the culture,which may be suitably monitored by an on-line optical probe. The term“turbidity”, as used herein, refers to the optical property that causeslight to be scattered and absorbed rather than transmitted in straightlines through the sample. Turbidity can be measured by commerciallyavailable turbidimeters. These turbidimeters usually measure the amountof light scattered at right angles to an incident light beam byparticles present in a fluid sample. In the present case, a turbidimeteris used to measure the light scattered by bacterial cells present in theculture medium at an angle of 90° relative to the incident light beam.In order to measure the cell density, which is defined herein as thenumber of Clostridium botulinum cells per unit volume of culture, theturbidimeter is calibrated with commercially available certifiedFormazin Turbidity Standards (i.e. defined particle suspensions). Themeasured turbidity values are expressed herein as Formazin TurbidityUnits (FTU).

In the context of the present invention, the fermentation is generallycontinued until the cell density of the culture, after it has beenincreased due to bacterial growth, decreases due to cell lysis. ForClostridium botulinum type A, in particular for the Hall strain, thecell density after 24 hours of cultivation is preferably about 1.3±0.3FTU. The pH after 24 hours is preferably about 5.7±0.2. At the end ofthe fermentation with Clostridium botulinum type A, in particular withthe Hall strain, the cell density is preferably below 0.8 FTU. The pH atthe end of the fermentation is usually about 5.5±0.3.

The fermentation time is, again for Clostridium botulinum type A and, inparticular, for the Hall strain, typically in the range of from 65 to 80hours and is, preferably, approximately 72 hours, e.g. 72 hours±4 hours,72 hours±2 hours, 72 hours±1 hour or 72 hours±0.5 hours. The culturevolume is not particularly limited but is typically in the range ofabout 10 to 40 liters, preferably about 20 liters. The yield of thebotulinum toxin complex after fermentation using a Clostridium botulinumtype A strain, in particular the Hall strain, is usually in the range of3.5±2.0 μg, in particular 3.5±1.0 μg, based on 1 ml of the fermentationmedium at the end of the fermentation.

Turbidity measurements, unlike transmitted light measurements that areconventionally used in the art for determining cell densities infermentation broths, are not device-specific and give more accurate and,in particular, much better reproducible and comparable measurementresults. Unexpectedly, it was found that the assessment of the celldensity by turbidity measurements is not only highly accurate andrepeatable, but also allows one to control the process so that formationof the single-chain form of the neurotoxic component is limited. Thus,the use of turbidity measurements for monitoring cell growth and, inparticular, for determining the end point of the fermentation, makes itpossible to decrease the amount of the unwanted single-chain form in theend product. This is a significant process improvement since currentpurification methods are not capable of separating the inactive(non-cleaved) single-chain form from the active (cleaved) two-chainform.

In accordance with the present invention, the Clostridium botulinumculture of step (a) is preferably obtained by (i) providing an initialculture of Clostridium botulinum having a cell density of 530 to 850FTU, particularly 600 to 800 FTU, more particularly 650 to 750 FTU, and(ii) adding a pre-determined amount of the initial culture to a culturemedium. Preferably, the initial culture is added to the fermentationmedium in an amount of from 5.0% to 10.0% v/v, preferably in an amountof from 7.7% to 8.2% v/v. It is further preferred that the initialculture has an anaerobic viable count of at least 5.0×10⁵ cfu/ml(colony-forming units/ml), particularly at least 2.0×10⁶ cfu/ml, moreparticularly more than 1.0×10⁷ cfu/ml, and most particularly from1.0×10⁷ cfu/ml to 1.0×10⁸ cfu/ml. Within the present invention, theaerobic or anaerobic viable count is determined by plating dilutionseries of an initial sample on blood agar plates, incubating the platesat a given temperature (e.g., 37° C.) and for a given time (e.g., 40hours to 72 hours) under aerobic or anaerobic conditions and countingthe colonies grown, particularly according to Pharm. Eur. 2.6.12 and USP<61>.

The initial culture may be obtained, for example, by first preparing apre-culture involving inoculation of a seed medium with Clostridiumbotulinum and growing the bacteria at a suitable growth temperature(e.g., 37° C.). An aliquot of the obtained pre-culture is then used forinoculation of a culture medium, followed by growing the bacteria at asuitable growth temperature. Next, an aliquot of the obtainedpre-initial culture is used to inoculate a culture medium at a suitablegrowth temperature until the desired cell density is reached. Then, analiquot of the obtained initial culture is used for inoculation of thefermentation medium used in step (a) of the process of the presentinvention.

The source of the Clostridium botulinum strain (e.g., the Hall strain)used within the present invention may be conveniently provided in formof a frozen aliquot of a working cell bank (WCB). The WCB is establishedfrom a master cell bank (MCB) of the respective strain according totechniques known in the art. The frozen aliquot of the WCB may, forexample, be provided in the form of a cryovial containing 800 μl of theWCB and 200 μl sterile glycerol as cryoprotectant. Typically, theanaerobic viable count of the frozen aliquot of Clostridium botulinum,in particular the Hall strain, is at least 5.0×10⁵ cfu/ml, preferablymore than 1.0×10⁷ cfu/ml. The frozen aliquots (e.g., cryovials) may bestored at −80° C. in a freezer or, preferably, at about −130° C. in thevapor phase of liquid nitrogen.

In step (b) of the process of the present invention, the neurotoxiccomponent is isolated from the produced botulinum toxin (complex).Processes for purification of the neurotoxic component from toxincomplexes produced by Clostridium botulinum are known in the art (see,e.g., DasGupta B. R. and Sathyamoorthy, V., Toxicon. 22:415-424, 1984;and WO 00/74703). The concentration of the purified neurotoxic componentat the end of the purification is typically in the range of 100 μg/ml to500 μg/ml, based on one ml of the final solution of the purifiedneurotoxic component.

A suitable isolation process for use within the present invention, inparticular for the isolation of the neurotoxic component of Clostridiumbotulinum toxin type A, including the Hall strain, includes the step ofacid precipitation of the botulinum toxin at the end of the fermentation(e.g., by adding 3 N sulfuric acid; final pH of about 3.5). Aftercentrifugation, the precipitate is extracted (e.g., with 0.2 M sodiumphosphate buffer, pH 6.0) to release the toxin complex into thesolution. The extract is then subjected to protamine sulfateprecipitation (e.g., 2% protamine sulfate) to precipitate nucleic acidsfrom the supernatant, and the toxin complex is precipitated from thesupernatant using ammonium sulfate (e.g., by adding 38 g ammoniumsulfate per 100 g of supernatant).

After solubilization with phosphate buffer (e.g., 50 mM sodiumphosphate, pH 6.0), the toxin is further purified by three ion exchangechromatographic steps in the following order: DEAE Sepharose Fast Flow,Q Sepharose Fast Flow and SP Sepharose Fast Flow. Following addition ofglycerol, the final eluate is filtered through a sterile filter, such asa 0.22 μm filter to obtain the final product. This final product afterpurification can then be further processed, e.g. supplemented withstabilizing aids (e.g., human serum albumin (HSA) or sucrose) and/orlyophilized.

In accordance with the present invention, the cultivating step (a) andthe isolating step (b) of the process of the present invention areconducted in a pressure gradient device. This device comprises a firstisolator unit and, optionally, a second or further isolator unit, aswell as a safety work bench. The safety work bench is used foraseptically loading the first isolator unit and/or second or furtherisolator unit with materials, in particular heat-sensitive materialswhich cannot be autoclaved (e.g., working cell banks). To this end, thematerial may be transferred from the safety work bench to theisolator(s) using a specific transfer system (e.g. an alpha/beta-portsystem available from the Getinge Group) as further described below.This is an important aspect of the present invention since it was foundto result in less contamination (microbial and particular impurities)and higher purity of the neurotoxic component of a botulinum toxin.

Within the present invention, the safety work bench is a Class II BSCprovided with a transfer system that allows for the aseptic transfer ofmaterial into and out of the BSC. A “biological safety cabinet” or“biosafety cabinet” or “BSC” within the meaning of the present inventionis an enclosed, ventilated laboratory workspace for protecting thelaboratory worker and the surrounding environment from risks ofhazardous agents, such as bacteria, viruses or any other toxic orpathogenic agents, and for maintaining the sterility of materials insidethe workspace. In other words, BSCs provide protection of experimentfrom ambient environment, and protection of ambient environment, fromexperiment. Within the context of the present invention, this alsoincludes the transfer of heat-sensitive material into isolator 1 orisolator 2 without a sterilization step, e.g. cell banks.

Preferably, the BSC used for the safety work bench is a Class II BSC,more preferably a Class II, Type A1 or Type A2 BSC, most preferably aClass II, Type A2 BSC, as classified by the U.S. Centers for DiseaseControl and Prevention (CDC) (see U.S. Department of Health and HumanServices, Public Health Service; Centers for Disease Control andPrevention; National Institutes of Health. Biosafety in Microbiologicaland Biomedical Laboratories. Appendix A—Primary Containment forBiohazards: Selection, Installation and Use of Biological SafetyCabinets. 5th Edition, HHS Publication No. (CDC) 21-1112, RevisedDecember 2009) and defined by NSF/ANSI Standard 49-2007 (see NSFInternational (NSF); American National Standards Institute (ANSI).NSF/ANSI Standard 49-2007. Class II (laminar flow) biosafety cabinetry.Ann Arbor (Mich.); 2004). The safety work bench is typically operated atthe same pressure as that of the production room.

A BSC comprises a work chamber, air supply means for supplying air of aunidirectional airflow traveling from an upper part to a lower part inthe work chamber, and air discharge means for discharging air of theunidirectional airflow. Biosafety cabinets work by drawing a curtain ofsterile air over the products that are being handled. The air is thendrawn underneath the work surface (e.g., table or bench), back to thetop. A part of the air is exhausted, while another part is againintroduced into the working space to draw a curtain of sterile air. Atsome point in the system, the air passes through one or more filters,usually a HEPA (class of high efficiency particulate air) filter, sothat both the exhaust air and the recirculation air are sterile andparticle-free. The exhaust air is made up by air that is drawn into thefront of the cabinet and then underneath the work surface to combinewith the recirculation air drawn from the cabinet inside underneath thework surface. In case of a typical Class II, Type A1 BSC, approximately30% of the air passes through an exhaust HEPA filter and approximately70% recirculates through the supply HEPA filter back into the work zoneof the cabinet. A Class II, Type A2 BSC is similar to the A1 Type, butthe minimum inflow velocity is typically about 100 ft/min or more.

It should be understood that a “BSC” within the meaning of the presentinvention is not a “clean bench”. A “clean bench”, as used herein,refers to a horizontal laminar flow “clean bench” or vertical flow“clean bench”, which generally only provide a Class 100 work area forprocedures requiring a particle-free environment. The make-up air isfiltered, while the exhaust air is not filtered. In contrast, both themake-up and exhaust air is filtered, e.g. HEPA-filtered, in case ofBSCs. Thus, a clean bench does only provide product protection but doesnot prevent the worker from being exposed to materials being manipulatedon the clean bench. Generally, clean benches are inappropriate for usewith any potentially biohazardous materials, including toxic, mutagenicor carcinogenic substances, biological toxins, infectious agents (e.g.,bacteria, viruses, parasites, etc.), and are generally not usable ifaseptic conditions are required for the work.

The transfer system of the safety work bench preferably comprises afirst transfer unit and a second transfer unit detachably connectable toeach other, wherein the first transfer unit is a sealable part mountedon a surface of the BSC, usually fixed to a wall of the BSC, and thesecond transfer unit is a sealable container. The sealable container maybe made of various materials, such as stainless steel, polyethylene andthe like. It may be rigid or flexible, e.g. in form of a bag, and may bea single-use or reusable container. Preferably, the transfer system isthe DPTE® transfer system (Getinge) comprised of two separate units,i.e. the Alpha and Beta parts, which are each fitted with a door, a lockand a sealing function and permit the successive transfer of materialswithout breaching integrity of the sterile or toxic environmentcontained within the Alpha or Beta component.

The pressure gradient device of the present invention further comprisesa first isolator unit and, optionally, a second or further isolatorunit. The first isolator unit and the second or further isolator unitare Class II BSCs provided with a transfer system that allows for theaseptic transfer of material into and out of the BSC. Generally, glovesare attached to the front to prevent contact with the neurotoxin. Thetransfer system may be, for example, the DPTE system described above inconnection with the safety work bench. The first isolator unit containsa fermenter in which the step of cultivating Clostridium botulinum isconducted. Both the first and second or further isolator units arelocated in the production room that is connected to the environment viaan air lock, wherein a pressure gradient is formed between the isolatorunit(s), the production room and the air lock.

Specifically, the pressure in the first and second or further isolatorunits is lower than that in the production room, for example 10 to 100Pa, preferably 20 to 80 Pa, more preferably 50 to 70 Pa, most preferably60 Pa lower than in the production room. Furthermore, while the pressurein the production room is higher than that in the first and second orfurther isolator units, it is still lower than ambient pressure, forexample 5 to 50 Pa, preferably 10 to 30 Pa, more preferably 12 to 18 Pa,and most preferably about 15 Pa lower than ambient pressure.

The pressure in the air lock is higher than ambient pressure, forexample 10 to 100 Pa, preferably 20 to 80 Pa, more preferably 25 to 35Pa, and most preferably 30 Pa higher than ambient pressure. In otherwords, there is typically a pressure difference between the air lock andthe production room of about 15 to 150 Pa, preferably about 30 to 110Pa, more preferably about 37 Pa to 53 Pa, and most preferably about 45Pa. The term “ambient pressure” means the pressure of the surroundingatmosphere and is generally about 1 atmosphere, but may vary dependingon geographical position or meteorological conditions.

The second or further isolator unit may be a BSC as that described abovein relation to the first isolator unit. It may be operated at the sameor different pressure as the first isolator unit. Furthermore, the firstisolator unit may not or may be connected with the second or furtherisolator units by, for example, one or more lockable air locks, doubletrap container or ports. In accordance with the present invention, thecultivating step (a) and the isolating step (b) may be conducted in thefirst isolator unit. Preferably, however, the cultivating step and atleast one stage of the purification step is conducted in the firstisolator unit, whereas the remaining purification stages are carried outin the second or further isolator units. The first isolator unit may beoperated at a higher temperature, e.g. at about 15° C. to 50° C. or 20°C. to 40° C., and the second or further isolator unit may be operated ata lower temperature, for example in the range of about −5° C. to +25° C.The term “about”, as used in the context of the present invention, means“approximately” or “nearly”. In the context of numerical values, withoutcommitting to a strict numerical definition, the term may be construedto estimate a value that is +/−10% of the value or range indicated.

The production room of the present invention is sealed and airtight roomof that can be operated at negative pressure. It can be accessed fromthe outside by one or more air locks. The production room contains thesafety work bench as well as the first isolator and, optionally, thesecond or further isolator. The supply of air into and out of theproduction room occurs preferably via filters, in particular HEPAfilters. Inside the production room, there is a controlled temperatureof, for example, 19° C. to 26° C., and a controlled relative humidityof, for example, 40% to 60%, particularly 55%. Certain operations oractivities, including measurements or tests, which are necessary for orrelated to the production of the botulinum toxin may be executed outsidean isolator unit, in particular if they are not associated with aerosolformation or if the biological active material is present in a formwhich does not pose any hazards for the persons engaged with botulinumtoxin production.

In another aspect, the present invention relates to a highly pureneurotoxic component of a Clostridium botulinum toxin having asingle-chain content of less than 2.00 wt. %. Preferably, thesingle-chain content is less than 1.90 wt. %, 1.80 wt. %, 1.70 wt. %,1.60 wt. %, 1.50 wt. %, 1.40 wt. %, 1.30 wt. %, 1.20 wt. %, 1.10 wt. %,1.00 wt. %, 0.90 wt. % or less than 0.80 wt. %. Furthermore, the highlypure neurotoxic component of the present invention has preferably atotal purity of at least 99.90 wt. %, more preferably of at least 99.95wt. %, and most preferably of 99.99 wt. %, wherein the term “totalpurity” has the meaning as defined above. In addition, the highly pureneurotoxic component of a Clostridium botulinum toxin of the presentinvention may have an endotoxin content of equal to or less than 5.0 IU,in particular equal to or less than 1.0 IU/ml, per ml of the finalproduct after purification, i.e. typically one ml of a solutioncontaining the highly pure neurotoxic component of a botulinum toxin ina concentration of 100 μg/ml to 500 μg/ml.

Furthermore, the highly pure neurotoxic component of a Clostridiumbotulinum toxin of the present invention has preferably a total aerobicviable cell count of less than 1 cfu/ml, preferably less than 0.5cfu/ml, more preferably 0 cfu/ml, based on one ml of a solutioncontaining the highly pure neurotoxic component of botulinum toxin in aconcentration of 100 μg/ml to 500 μg/ml.

Moreover, the highly pure neurotoxic component of a Clostridiumbotulinum toxin of the present invention has a biological activity(relative potency) in an LD₅₀ assay of 4.0 to 8.0 μg protein/LD₅₀ unit,particularly 5.0 to 6.0 μg protein/LD₅₀ unit. The LD₅₀ assay used hereinfor the assessment of the biological activity is known in the art anddescribed, for example, in Pearce L. B., Borodic G. E., First E. R., andMacCallum R. D., Measurement of botulinum toxin activity: evaluation ofthe lethality assay, Toxicol. Appl. Pharmacol. 128:69-77, 1994. Thebiological activity is expressed in “units” (U), wherein 1 U is definedas being the equivalent amount of toxin (i.e. the neurotoxic component)that kills 50% of a specified mouse population after intraperitonealinjection.

The above described highly pure neurotoxic component of a Clostridiumbotulinum toxin can be obtained by the process according to theinvention. Thus, in a preferred embodiment of the present invention, thehighly pure neurotoxic component of a Clostridium botulinum toxindescribed herein is prepared by the process according to the invention.

In a further aspect, the present invention relates to a pharmaceuticalcomposition comprising a highly pure neurotoxic component of aClostridium botulinum toxin and one or more pharmaceutically acceptablecarriers. A “pharmaceutical composition” is a formulation in which anactive ingredient is contained or comprised. The dosage form of thepharmaceutical composition is not particularly limited but is preferablya parenteral formulation, such as an aqueous or non-aqueous solution ordispersion for injection or infusion. The pharmaceutical composition ofthe present invention may be lyophilized or vacuum dried, reconstitutedor in solution. When reconstituted, it is preferred that thereconstituted solution is prepared by adding sterile physiologicalsaline (i.e., 0.9 wt. % NaCl).

The pharmaceutical composition generally includes an effective amount ofthe neurotoxic component of the present invention. Within the presentinvention, the term “effective amount” refers to an amount of neurotoxiccomponent which, after administration, results in a partial or completeremoval of disease symptoms or conditions. A therapeutically effectiveamount can be administered in one or more administrations, applicationsor dosages and is not intended to be limited to a particular formulationor administration route. Effective amounts are generally in the range of1 to 2000 U. However, also doses of up to 5000 U may be used. When highdoses of the neurotoxic component are to be administered to a subject,it may be beneficial to split the treatment into more than one treatmentsession. The term “more than one treatment session” means, e.g., 2, 3,4, 5, 6, 7, 8, 9 or 10 treatment sessions.

Within the context of the present invention, a “carrier” refers to adiluent or vehicle whereby the active ingredient is administered.Suitable carriers for use herein include sterile liquids or dispersions,especially those suited for parenteral administration (e.g., byintramuscular or subcutaneous injection), as discussed in Remington: TheScience and Practice of Pharmacy, 20th Edition (2000). Preferably, thecarrier is water or an aqueous pH buffer, such as a phosphate buffer,phosphate buffered saline (PBS) or an acetate buffer. The term“pharmaceutically acceptable”, as used herein, refers to those compoundsor substances which are, within the scope of sound medical judgment,suitable for contact with the tissues of mammals, especially humans,without excessive toxicity, irritation, allergic response and otherproblem complications.

In addition, the pharmaceutical composition of the present invention mayinclude additional components, such as excipients, stabilizers and/orcryoprotectants. The excipients may include, but are not limited to,sugars (e.g., mono- or disaccharides like sucrose), salts (e.g., NaCl),detergents (e.g., non-ionic, anionic or cationic surfactants), andchelating agents (e.g., EDTA). Examples of stabilizers includeproteinaceous stabilizers, for example gelatin or albumin (i.e. HSA),and non-proteinaceous stabilizers, for example hyaluronic acid,polyvinylpyrrolidone, polyethyleneglycol and mixtures thereof. Thecryoprotectants exert a stabilizing effect on proteins (i.e. theneurotoxic component) during lyophilization and include, inter alia,alcohols, especially polyalcohols like inositol, mannitol or glycerol.Also, the pharmaceutical composition may include one or more additionalactive substances that are co-administered with the neurotoxic componentof the present invention.

Preferably, the pharmaceutical composition of the present inventioncomprises a highly pure neurotoxic component as described herein, a pHbuffer, preferably a phosphate buffer or an acetate buffer, and ahyaluronic acid stabilizer or a polyvinylpyrrolidone stabilizer or apolyethyleneglycol stabilizer or a BSA or HSA stabilizer. Additionally,the pharmaceutical composition may comprise a polyalcohol and/or adetergent.

In yet a further aspect, the present invention relates to the use of aneurotoxic component of a Clostridium botulinum toxin of the presentinvention as a medicament. A “medicament”, as used herein, refers to anycomposition comprising a neurotoxic component of a Clostridium botulinumtoxin for the treatment of a disease. In this context, the term“disease” is not limited to a particular disease, but includes anydisorder or condition that disrupts body functions, systems or organs.

In still another aspect, the present invention relates to the use of aneurotoxic component of a Clostridium botulinum toxin of the presentinvention for the treatment of a disease or condition associated withhyperactive cholinergic innervation of muscles or exocrine glands. Theterm “treatment”, as used herein, includes therapeutic treatment andprophylactic treatment (prevention) as well as cosmetic treatment of adisease or condition. A “treatment” within the meaning of the presentinvention generally involves the administration of an effective amountof highly pure Clostridium botulinum neurotoxic component of the presentinvention to a subject having the disease or condition associated withhyperactive cholinergic innervation of muscles or exocrine glands.

The term “subject”, as used herein, refers to a mammal, preferably ahuman. The subject may have never been exposed to botulinum toxin, ormay have been exposed to botulinum toxin. The term “effective amount”,as used in this context, has the same meaning as described above inconnection with the pharmaceutical composition.

The term “hyperactive cholinergic innervation”, as used herein, relatesto a synapse, which is characterized by an unusually high amount ofacetylcholine release into the synaptic cleft. “Unusually high” relatesto an increase of up to 25%, up to 50% or more with respect to areference activity which may be obtained, for example, by comparing therelease with the release at a synapse of the same type, but which is notin a hyperactive state, wherein muscle dystonia may be indicative of thehyperactive state. “Up to 25%” means, for example, about 1% to about25%. Methods for performing the required measurements are known in theart.

Exemplary diseases or conditions associated with hyperactive cholinergicinnervation of muscles or exocrine glands are described in detail inDressler, D., Botulinum Toxin Therapy, Thieme, Stuttgart, N.Y., 2000,and include, but are not limited to, dystonia (e.g., cranial dystonia,cervical dystonia, pharyngeal dystonia, laryngeal dystonia, limbdystonia), cosmetic use (e.g., craw's feet, frowning, facialasymmetries, mentalis dimples), strabism, exocrine gland hyperactivity(e.g., Frey syndrome, Crocodile Tears syndrome, hyperhidrosis),rhinorrhea, hypersalivation (drooling), spastic conditions, and otherdiseases.

Other diseases associated with hyperactive cholinergic innervation ofmuscles or exocrine glands include, for example, palatal myoclonus,myoclonus, myokymia, rigidity, benign muscle cramps, hereditary chintrembling, paradoxic jaw muscle activity, hemimasticatory spasms,hypertrophic branchial myopathy, maseteric hypertrophy, tibialisanterior hypertrophy, nystagmus, oscillopsia, supranuclear gaze palsy,epilepsia partialis continua, planning of spasmodic torticollisoperation, abductor vocal cord paralysis, recalcitant mutationaldysphonia, upper oesophageal sphincter dysfunction, vocal foldgranuloma, stuttering, Gilles de la Tourette syndrome, middle earmyoclonus, protective larynx closure, postlaryngectomy speech failure,protective ptosis, entropion, sphincter Odii dysfunction,pseudoachalasia, nonachalsia oesophageal motor disorders, vaginismus,postoperative immobilization, tremor, bladder dysfunction, hemifacialspasm, reinnervation dyskinesias, stiff person syndrome, tetanus,prostate hyperplasia, adipositas treatment, infantile cerebral palsy,achalasia and anal fissures.

Suitable administration routes include, but are not limited to,parenteral administration, in particular subcutaneous and intramuscularinjection. The administration regimen is not particularly limited andincludes, for example, bi-weekly, monthly, once every other month, onceevery third, sixth or ninth month and once-a-year or single applicationadministration schemes. The therapeutically effective dose of the highlypure neurotoxic component of the present invention that is administeredto the subject depends on the mode of application, the type of diseaseor condition, the subject's weight, age, sex and state of health, andthe like. Administration can be single or multiple, as required. Thehighly pure neurotoxic component of the present invention may also beco-administered with other active substances.

The present invention will now be further illustrated by the following,non-limiting examples.

EXAMPLES

The following examples show that the use of a pressure gradient deviceas described herein permits the creation of working zones that areaseptic and have an extremely low number of airborne particles. This hassurprisingly been found to have a strong impact on the overall productquality, in particular with respect to purity of the neurotoxiccomponent.

Example 1 Construction of a Safety Work Bench

The “Alpha” part of a DPTE® transfer system (Getinge Group) was mountedon the right side wall of a HERAsafe® (NSF) Class II, Type A2 biologicalsafety cabinet equipped with HEPA filters (Thermo Fisher Scientific,Inc.). A rigid transfer container (DPTE 190 Beta container made ofstainless steel) was used as the movable DPTE® “Beta” part. The twoseparate units of the DPTE® transfer system, i.e. the Alpha and Betaparts, are each fitted with a door, a lock and a sealing function andpermit the successive transfer of toxic or pathogenic materials withoutbreaching integrity of the sterile or toxic environment contained withinthe Alpha or Beta component.

In all experiments described below, this safety workbench was used andlocated in a production room (operated at neutral pressure relative tothat of the production room). The production room was operated at atemperature of 19° C. to 26° C. and a pressure of −15 Pa relative toambient pressure. In the production room, there were further twoisolators (isolator 1: fermentation; isolator 2: purification) operatedat a pressure of −60 Pa relative to the pressure in the production room.The production room was connected to the environment by a personnel airlock and a material air lock.

Example 2 Microbial Purity in the Isolator Unit of the Present Invention

The aim of this study was to show that cultivation of Clostridiumbotulinum using the process of the invention is carried out underaseptic conditions. Therefore all cultivation procedures were simulatedwithout microorganisms in three consecutive runs. All culture media andadditives were replaced by media for sterility testing (TSB- andthioglycolate medium for aerob or anaerob process conditions).

To verify the absence of microbiological contamination, samples takenfrom the different process steps were incubated for at least 14 days,but not exceeding 21 days, at 20° C. to 35° C. as follows: incubation at20° C. to 25° C. for at least 7 days, directly followed by incubation at30° C. to 35° C. for at least 7 days.

As can be seen from Table 1, no microbial contaminations could bedetected throughout the different cultivation stages. This demonstratesthat the isolator used within the present invention—despite beingoperated at lower pressure than that of the production room, whichresults in the influx of potential impurities—allows for aseptic processcontrol.

TABLE 1 Microbial contamination in the isolator unit Experiment 1Experiment 2 Experiment 3 Process stage of (aerob) (aerob) (anaerob)fermentation Microbial contaminations Preculture none none none Initialculture 1 none none none Glucose solution none none none Initial culture2 none none none Fermentation culture none none none 96 h Fermentationculture none none none 120 h

In addition, the number of airborne germs was determined at threedifferent measuring points (designated MP18, MP19 and MP20) in theisolator unit 1. As shown in Table 2, no airborne germs could bedetected at all three measuring points. In contrast, the average numberof airborne germs in the production room, as measured in 2010, was foundto be 3 cfu/m³.

TABLE 2 Number of airborne germs in the isolator unit Measuring point inthe isolator unit MP18 MP19 MP20 MP18 MP19 MP20 Lot. Airborne germs[cfu/m³] MP050807 0 0 0 0 0 0 MP050908 0 0 0 0 0 0 MP051009 0 0 0 0 0 0MP061201 0 0 0 0 0 0 MP071007 0 0 0 0 0 0 MP090201 0 0 0 0 0 0 MP0910020 0 0 0 0 0 MP110504 0 0 0 0 0 0

Thus, the use of an isolator unit as employed in the process of thepresent invention allows the creation of working zones that are free ofmicrobial contamination and airborne germs.

Example 3 Particle Content in the Isolator Unit

Another important factor, in addition to microbial contamination andnumber of airborne germs, which determines product quality is theconcentration of particulate impurities. This is of particularsignificance in the pharmaceutical industry, where the protection of agiven product from (airborne) particles is critically important.Therefore, the number of airborne particles in different working zones(designated MP18, MP19, and MP20) of the isolator unit used within thepresent invention was measured. The particle measurement was conductedby using a particle counter commercially available from CASClean-Air-Service AG, Switzerland. The results are shown in Table 3below.

TABLE 3 Number of airborne particles Particles ≥0.5 μm [ft⁻³] Particles≥5.0 μm [ft⁻³] Measuring point in the isolator Lot No. MP18 MP19 MP20MP18 MP19 MP20 MP050807 5 5 8 0 0 0 3 15 18 1 1 1 MP050908 5 2 7 3 1 2 713 1 3 4 0 MP051009 0 5 1 0 1 0 10 6 3 1 2 0 MP061201 1 2 3 0 0 1 5 6 51 1 1 MP071007 1 0 0 0 0 0 2 14 3 1 0 2 MP090201 0 5 0 0 3 0 3 6 7 0 0 1MP091002 0 0 0 0 0 0 0 11 3 0 2 1 MP110504 2 1 2 0 0 0 3 1 1 2 0 0

The results show that the isolator unit used within the presentinvention contains only very few particles with a diameter ≥0.5 μm andessentially no particles with a diameter ≥5.0 μm at all three measuringpoints. In contrast, the average particle numbers in the productionroom, as measured in 2010, was found to be 434 per ft³ for particleswith diameters ≥0.5 μm, and 22 per ft³ for particles with diameters ≥5.0μm. This demonstrates the very high particulate purity in the isolatorunit of the present invention compared to that in the surroundingproduction room.

Example 4 Product Quality in Terms of Biochemical Purity

The total purity and single-chain content of different lots ofneurotoxic components purified from Clostridium botulinum toxin type Aproduced by the Hall strain (ATCC 3502) with use and without use of theisolator of the present invention were determined.

A volume of 10 ml culture medium was inoculated with an aliquot of aWorking Cell Bank of Clostridium botulinum type A Hall strain (ATCC3502) to obtain a preculture of a total volume of 10 ml. An aliquot ofthe preculture was used to inoculate 50 ml medium to obtain InitialCulture 1. Initial Culture 2 was then obtained by inoculating 1600 mlmedium with an aliquot of Initial Culture 1. The medium of thefermentation culture (17.4 l) was sterilized in situ in the fermenter.After adding 1 l of sterile glucose solution, the fermentation cultureis inoculated with 1600 ml of Initial Culture 2. Fermentation wascarried out at 33.5° C. for 72 h.

The purification was performed out as described hereinabove. “Totalpurity” is defined as the weight percentage of the single-chain andtwo-chain forms of the neurotoxic component, based on the total weightof a sample of the purified neurotoxic component. The results obtainedare shown in Table 4 below.

TABLE 4 Total purity of the neurotoxic component of Clostridiumbotulinum toxin type A Prepared without isolator Prepared with isolatorLot No. Total purity [%] Lot. No. Total purity [%] A 29-02 98.8 MP050403100.0 A 30-02 97.0 MP050606 100.0 A 31-02 97.6 MP050807 100.0 Mean value97.8 MP050908 99.0 MP051009 100.0 MP101003 100.0 MP110101 100.0 MP110302100.0 Mean value 99.9

As can be seen from Table 4, the total purity of the neurotoxiccomponent prepared according to the present invention is as high as 99.9wt. %. In comparison, the total purity obtained without using theisolator technology of the present invention is only 97.8 wt. %. Inother words, the neurotoxic component formulation prepared according tothe present invention consists essentially only of the neurotoxiccomponent (in single-chain or two-chain form), whereas the neurotoxiccomponent formulation prepared without using the isolator technology ofthe present invention contains a significant amount of contamination of2.2 wt. %.

The lots of Table 4 were further evaluated with regard to their contentof the single-chain form of the neurotoxic component. SDS-PAGE withphotometric evaluation was used to determine the single-chain content.The single-chain content is an especially important parameter since theuncleaved single-chain form is essentially inactive and, thus, an“impurity” within the meaning of the present invention. Furthermore, itcannot be chromatographically resolved from the active two-chain form.The results are shown in Table 5.

TABLE 5 Content of the single-chain form of the neurotoxic componentPrepared without isolator Prepared with isolator Single-chainSingle-chain Lot No. content [wt. %] Lot. No. content [wt. %] A 29-021.8 MP050403 0.4 A 30-02 1.9 MP050606 0.8 A 31-02 1.4 MP050807 2.0 Meanvalue 1.7 MP050908 1.3 MP051009 1.1 MP101003 1.6 MP110101 1.3 MP1103021.2 Mean value 1.2

The results unexpectedly show that the process of the present inventionprovides a mean single-chain content of 1.2 wt. % compared to the 1.7wt. % obtained for the comparative lots.

Example 5 Product Quality in Terms of Microbial Purity

The microbial purity (bioburden) of the product was assessed bymeasuring the aerobic microbial bioburden according to Pharm. Eur.2.6.12 and USP <61> at different process stages of lots preparedaccording to the present invention in comparison to lots preparedwithout the isolator technology used in the present invention. Theresults are shown in Tables 6, 7, and 8.

TABLE 6 Aerobic bioburden at early process stage “after dialysis 1”Prepared without isolator Prepared with isolator Total aerobic Totalaerobic Lot No. count [cfu/ml] Lot. No. count [cfu/ml] A 29-02 220MP050807 <10 A 30-02 440 MP050908 <10 A 31-02 980 MP051009 <10 Meanvalue 547 MP101003 <5 MP110101 <5 MP110302 <5 Mean value <8

TABLE 7 Aerobic bioburden at late process stage “SP-Pool” Preparedwithout isolator Prepared with isolator Total aerobic Total aerobic LotNo. count [cfu/ml] Lot. No. count [cfu/ml] A 29-02 28 MP050807 <10 A30-02 52 MP050908 <10 A 31-02 34 MP051009 <10 Mean value 38 MP101003 <5MP110101 <5 MP110302 <5 Mean value <8

TABLE 8 Aerobic bioburden of the final product obtained at the end ofthe purification process Prepared without isolator Prepared withisolator Total aerobic Total aerobic Lot No. count [cfu/ml]¹ Lot No.count [cfu/ml]¹ A 29-02 <2 MP101003 0 A 30-02 <2 MP110101 0 A 31-02 <2MP110302 0 Mean value <2 Mean value 0 ¹= based on one ml of the finalproduct (solution containing 100-500 μg/ml highly purified neurotoxiccomponent

The results presented in Tables 6, 7 and 8 show that the bioburden at anearly process stage (after dialysis 1) of the process according to thepresent invention is <8 and, thus, much lower than the mean value of 547obtained without using an isolator of the present invention. Thisensures that there is no aerobic (bacterial) count in the final product.

Example 6 Product Quality in Relation to Endotoxin Contamination

The endotoxin level of lots prepared according to the present inventionwas measured according to Pharm. Eur. 2.6.14 and USP <85> and comparedwith that determined for lots prepared without using the isolatortechnology of the present invention. The endotoxin values measured areindicated in IU per ml of the final product (solution containing 100-500μg/ml of highly purified neurotoxic component). The results are shown inTable 9.

TABLE 9 Endotoxin load of the final product Prepared without isolatorPrepared with isolator Endotoxin Endotoxin Lot No. content [IU/ml] Lot.No. content [IU/ml] A 29-02 <2.0 MP050403 <1.0 A 30-02 <1.5 MP050606<0.8 A 31-02 <1.5 MP050807 <0.8 Mean value <1.7 MP050908 <0.8 MP051009<0.8 MP101003 <0.8 MP110101 <0.8 MP110302 <0.8 Mean value <0.8

As can be seen from Table 9, the endotoxin levels of lots preparedaccording to the present invention are significantly lower than that ofthe comparative lots. The mean endotoxin level of <0.8 IU/ml found forthe inventive neurotoxic component is an acceptable endotoxin level fora botulinum toxin-containing pharmaceutical product.

The above results show that the process of the present invention allowsfor the preparation of a highly pure neurotoxic component of a botulinumtoxin while at the same time meeting the standards on occupationalhealth and safety.

The invention claimed is:
 1. A highly pure neurotoxic component of aClostridium botulinum toxin having a single-chain content of less than2.00 wt. %, obtainable by a process comprising: (a) cultivatingClostridium botulinum under conditions that allow production of abotulinum toxin, and (b) isolating the neurotoxic component from thebotulinum toxin, wherein cultivating (a) and isolating (b) are conductedin a pressure gradient device, the pressure gradient device comprising afirst isolator unit containing a fermenter for cultivating Clostridiumbotulinum and a safety work bench, and optionally a second or furtherisolator unit, wherein the first and second or further isolator unitsare located in a production room that is connected to the environmentvia an air lock, the pressure in the first and second or furtherisolator units being lower than that in the production room, thepressure in the production room being lower than ambient pressure, andthe pressure in the air lock being higher than ambient pressure, andwherein the safety work bench is located in the production room and is aClass II BSC (Biological Safety Cabinet) provided with a transfer systemthat allows for the aseptic transfer of material into and out of theBSC.
 2. The highly pure neurotoxic component of claim 1, whereincultivating temperature is maintained at a temperature of 33.5° C.±1.0°C.
 3. The highly pure neurotoxic component of claim 1, wherein celldensity during cultivating (a) is monitored by turbidity measurementsusing formazin as a primary turbidity standard.
 4. The highly pureneurotoxic component of claim 1, wherein cultivating Clostridiumbotulinum is continued until cell density decreases from a cell densityof 1.3±0.3 FTU reached after 24 hours of cultivation to less than 0.8FTU.
 5. The highly pure neurotoxic component of claim 1, whereinClostridium botulinum culture used in (a) is obtained by (i) providingan initial culture of Clostridium botulinum having a cell density of 530to 850 FTU and (ii) adding a pre-determined amount of initial culture toa fermentation medium.
 6. The highly pure neurotoxic component of claim5, wherein the initial culture is added to the fermentation medium in anamount of from 5.0% to 10.0% v/v.
 7. The highly pure neurotoxiccomponent of claim 1, wherein the transfer system of the Class II BSCcomprises a first transfer unit and a second transfer unit detachablyconnectable to each other, the first transfer unit being a sealable partmounted on a surface, or fixed to a wall, of the BSC, and the secondtransfer unit being a sealable container.
 8. The highly pure neurotoxiccomponent of claim 1, wherein the Clostridium botulinum is Clostridiumbotulinum type A, including the Hall strain (ATCC 3502).
 9. The highlypure neurotoxic component of claim 1, further having a total purity ofat least 99.90 wt. %.
 10. The highly pure neurotoxic component of claim1, further having an endotoxin content of 5.0 IU/ml or less and/or atotal anaerobic viable count of less than 1 cfu/ml.
 11. A pharmaceuticalcomposition comprising a highly pure neurotoxic component of aClostridium botulinum toxin according to claim 1 and one or morepharmaceutically acceptable carriers.
 12. A medicament comprising ahighly pure neurotoxic component of a Clostridium botulinum toxinaccording to claim
 1. 13. A method for treatment of a disease orcondition associated with hyperactive cholinergic innervation of musclesor exocrine glands comprising employing a highly pure neurotoxiccomponent of a Clostridium botulinum toxin according to claim
 1. 14. Amethod of making the highly pure neurotoxic component of claim 1,comprising: (a) cultivating Clostridium botulinum under conditions thatallow production of a botulinum toxin, and (b) isolating the neurotoxiccomponent from the botulinum toxin, wherein cultivating (a) andisolating (b) are conducted in a pressure gradient device, the pressuregradient device comprising a first isolator unit containing a fermenterfor cultivating Clostridium botulinum and a safety work bench, andoptionally a second or further isolator unit, wherein the first andsecond or further isolator units are located in a production room thatis connected to the environment via an air lock, the pressure in thefirst and second or further isolator units being lower than that in theproduction room, the pressure in the production room being lower thanambient pressure, and the pressure in the air lock being higher thanambient pressure, and wherein the safety work bench is located in theproduction room and is a Class II BSC (Biological Safety Cabinet)provided with a transfer system that allows for the aseptic transfer ofmaterial into and out of the BSC.
 15. The method according to claim 14,wherein cultivating temperature is maintained at a temperature of 33.5°C.±1.0° C.
 16. The method according to claim 14, wherein cell densityduring cultivating (a) is monitored by turbidity measurements usingformazin as a primary turbidity standard.
 17. The method according toclaim 14, wherein cultivating Clostridium botulinum is continued untilcell density decreases from a cell density of 1.3±0.3 FTU reached after24 hours of cultivation to less than 0.8 FTU.
 18. The method accordingto claim 14, further comprising in (a): (i) providing an initial cultureof Clostridium botulinum having a cell density of 530 to 850 FTU and(ii) adding a pre-determined amount of initial culture to a fermentationmedium.
 19. The method according to claim 18, wherein initial culture isadded to the fermentation medium in an amount of from 5.0% to 10.0% v/v.20. The method according to claim 14, wherein the highly pure neurotoxiccomponent of claim has a total purity of at least 99.90 wt. %.